The presence in synaptic membranes of high affinity recognition sites for benzodiazepines capable of eliciting biochemical, physiological or behavioral responses when acted upon by specific ligands, prompted the search for an endogenous ligand that operates in physiological conditions. The existence of an endogenous modulator for the benzodiazepine-GABA receptor complex was suggested by an early observation that the KD for 3H-diazepam binding to GABA-free crude synaptic membranes was reduced by approximately 50% by repeated washes of the membranes with small concentrations of detergents. It was soon discovered that the membrane extract contained a peptide which produced a dose-related increase of KD of diazepam binding without affecting the Bmax. Recently we developed a relatively simple procedure to isolate and purify this peptide to homogeneity. The peptide was termed DBI (diazepam binding inhibitor) and its extraction was routinely carried out using as starting material rat brain homogenized in hot (80C) 1 N acetic acid. Purification was achieved using Sephadex G-100 and G-75 chromatography and reverse phase HPLC. Studies with the antibody reveal that DBI is unevenly distributed in brain and behavioral studies indicate that DBI acts like beta-carboline in facilitating punished behavior.